Colloidal gold is a dispersion of gold nanoparticles in a colloidal suspension liquid, typically water but other liquids can also be used as discussed below. Gold nanoparticles (Au NPs) have attracted substantial interest from scientists for over a century because of their unique physical, chemical, and surface properties, such as: (i) size- and shape-dependent strong optical extinction and scattering which is tunable from ultraviolate (UV) wavelengths all the way to near infrared (NIR) wavelengths; (ii) large surface areas for conjugation to functional ligands; and (iii) little or no long-term toxicity or other adverse effects in vivo allowing their high acceptance level in living systems. Gold nanoparticles are now being widely investigated for their potential use in a wide variety of biological and medical applications as imaging contrast agents (Nat. Biotechnol. 2008, 26, 83 and Nano Lett. 2005, 5, 829), therapeutic agents (Nano Lett. 2007, 7, 1929 and Sci. Transl. Med. 2010, 2), biological sensors (Chem. Soc. Rev. 2008, 37, 2028), and cell-targeting vectors (Nano Lett. 2007, 7, 247).
Currently, the overwhelming majority of gold nanoparticles are prepared by using the standard wet chemical sodium citrate reduction of tetrachloroaurate (HAuCl4) methodology. This method results in the synthesis of spherical gold nanoparticles with diameters ranging from 5 to 200 nanometers (nm) which are capped or covered with negatively charged citrate ions, which prevents the nanoparticles from aggregating by providing electrostatic repulsion.
Other wet chemical methods for formation of gold nanoparticles include the Brust method, the Perrault method and the Martin method. The Brust method relies on reaction of chlorauric acid with tetraoctylammonium bromide in toluene and sodium borohydride. The Perrault method uses hydroquinone to reduce the HAuCl4 in a solution containing gold nanoparticle seeds. The Martin method uses reduction of HAuCl4 in water by NaBH4 wherein the stabilizing agents HCl and NaOH are present in a precise ratio. All of the wet chemical methods rely on first converting gold (Au) with strong acid into the atomic formula HAuCl4 and then using this atomic form to build up the nanoparticles in a bottom-up type of process. All of the methods require the presence of stabilizing agents to prevent the gold nanoparticles from aggregating and precipitating out of solution.
On the other hand, over the past few decades, a physical method of making gold nanoparticles based on pulsed laser ablation of a gold target immersed in a liquid has been attracting increasingly widespread interest. In contrast to the chemical procedures, pulsed laser ablation of a gold target immersed in a liquid offers the possibility of generating stable gold nanocolloids while avoiding chemical precursors, reducing agents, and stabilizing ligands, all of which could be problematic for the subsequent functionalization and stabilization of the nanoparticles. Therefore, since it was pioneered by Henglein and Fojtik for preparing nano-size particles in either organic solvents or aqueous solutions as well as by Cotton for preparation of water-borne surface-enhanced Raman scattering active metallic nanoparticles with bare surfaces in 1993, the application of pulsed laser ablation of metal targets in liquids has gained much interest and has evolved as one of the most important physical method for obtaining colloidal gold nanoparticles, especially after the advent of femtosecond lasers, which are capable of eliminating some problems associated with the use of nanosecond lasers. Compared to laser ablation with pulses of longer duration, e.g. nanoseconds, the irradiation of metal targets by femtosecond laser pulses offers a precise laser-induced breakdown threshold and can effectively minimize the heat affected zones since the femtosecond laser pulses release energy to electrons in the target on a time-scale much faster than electron-phonon thermalization processes.
For most practical biomedical applications of gold nanoparticles, chemical stability in biological medium, biocompatibility, and targeting efficacy are the key requirements. Surface modifications are essential for meeting these requirements since interactions of gold nanoparticles with complex biological environments and biomolecules both on the surface of and inside the cells highly depend on the chemical nature of their solvent-accessible surface.
PEGylation, coating surface of gold nanoparticles with poly(ethylene glycol) (PEG) molecules, is the most commonly used surface modification approach to optimize the surface properties and functionalities of gold nanoparticles. For instance, a layer of PEG on the surface of gold nanoparticles enhances their solubility and stability under physiological conditions by providing a steric barrier. Also, when heterobifunctional PEG derivatives having amine (—NH2) or carboxyl (—COOH) groups are incorporated onto surface of gold nanoparticles, these functional groups enable additional covalent surface modification with targeting ligands via conventional carbodiimide coupling chemistry (Nat. Biotechnol. 2008, 26, 83, J. Phys. Chem. C 2008, 112, 8127, J. Am. Chem. Soc. 2007, 129, 2871, and ACS Nano 2010, 4, 5887), which provides a route to further functionalization to generate targeting nanoparticles (Langmuir 2007, 23, 5352, Langmuir 2006, 22, 11022, Nano Lett. 2005, 5, 473, Chem. Commun. 2007, 4580, Langmuir 2007, 23, 7491, Small 2011, 7, 2412, and Nanoscale Res. Lett. 2011, 6).
Traditionally, surface modification of gold nanoparticles with PEG containing reactive functional groups, such as —COOH and —NH2, requires a large excess amount of PEG, sometimes over a 10 fold excess, to prevent aggregation of the gold nanoparticles. It is undesirable to have the excess unreacted free PEG molecules left in the gold nanocolloids since it might interfere with or alter the expected functionalities of the gold nanoparticle conjugates formed. It is not easy, however, to remove the excess free ligand without inducing aggregation or leading to a noticeable loss of gold nanoparticle conjugates. Furthermore, because the PEG molecules must be added in such a large excess, it is not possible to prepare gold nanoparticles either with a defined number of PEG molecules per nanoparticle, which would be very beneficial for many applications and fundamental studies, or with multiple different types of PEG molecules with predetermined ratio. Finally, current surface modification of gold nanoparticles with PEG containing functional groups, such as —COOH and —NH2, by adding to solution of gold nanoparticles a large excess of PEG molecules often results in highly charged surfaces, which promote strong non-specific binding to various cells and tissues. Consequently, after systemic administration these gold nanoparticles are rapidly cleared from the blood stream by the reticuloendothelial system (RES) and the mononuclear phagocytic system (MPS) in the liver, spleen, and bone marrow, resulting in reduced bioavailability of the targeting agents, a low therapeutic index and potential toxicity to healthy organs. Therefore, a technique granting the ability to control density of functional PEG molecules on surface of gold nanoparticles would have profound implications in biomedicine, for instance minimizing their macrophage recognition via optimizing surface charge (or zeta potential) of gold nanoparticle by controlling the ratio between the number of negative charged PEG molecules (such as PEG molecule terminated with COOH group at its distal end) and the number of positive charged PEG molecules (such as PEG molecule terminated with NH2 group at its distal end) bound on their surface.